Molecules 2011, 16, 5938-5962; doi:10.3390/molecules16075938
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Review
In Mesopore Protein Digestion:
A New Forthcoming Strategy in Proteomics
Rocco Savino, Francesca Casadonte and Rosa Terracciano *
Laboratory of Mass Spectrometry and Proteomics, Department of Experimental and Clinical Medicine,
“Magna Græcia” University of Catanzaro, Catanzaro 88100, Italy
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +39-0961-3694085; Fax: +39-0961-3694090.
Received: 2 June 2011; in revised form: 7 July 2011 / Accepted: 13 July 2011 /
Published: 15 July 2011
Abstract: The conventional protocols for in solution or in gel protein digestion require
many steps and long reaction times. The use of trypsin immobilized onto solid supports has
recently captured the attention of many research groups, because these systems can speed-up
protein digestion significantly. The utilization of new materials such as mesoporous silica
as supports, in which enzyme and substrate are dramatically concentrated and confined in
the nanospace, offers new opportunities to reduce the complexity of proteomics workflows.
An overview of the procedures for in situ proteolysis of single proteins or complex protein
mixtures is reported, with a special focus on porous materials used as catalysts. The
challenging efforts for designing such systems aimed at mimicking the biochemistry of
living cells are reviewed. Potentials, limitations and challenges of this branch of enzyme
catalysis, which we indicate as in mesopore digestion, are discussed, in relation to its
suitability for high-speed and high-throughput proteomics.
Keywords: mesoporous materials; protein digestion; catalysis; mass spectrometry; proteomics;
nanotechnology
1. Introduction
Proteomic approaches have been increasingly applied to the study of protein networks and
expression patterns in cells, tissues, and organisms, exploring their function at the molecular level.
Over the last few years, proteomic research has stimulated the application of new techniques and
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experimental strategies, expanding its interface role to broad and diverse research areas of science and
technology [1-4]. Mass spectrometry (MS) is considered the gold standard for the wide ranging
applications of proteomics, becoming the method of choice for protein analysis and identification [5].
Among different possible strategies, two main prototypic mass-based workflows are mostly pursued in
proteomics: Differential Proteomics and Shotgun Proteomics. In the case of Differential Proteomics
the workflow involves the separation of proteins by the traditional 1- or 2-D gel electrophoresis (GE),
followed by proteolytic digestion of protein band/spots of interest; the resulting digested mixture is
then analyzed by MS for protein identification. Shotgun Proteomics is based on enzymatic digestion of
complex protein mixtures, followed by extensive fractionation of peptides by multidimensional liquid
chromatography (LC) and automated MS/MS analysis [6,7]. In this two proteomic workflows,
proteolysis is performed according to two different protocols: in gel digestion [8,9] in the former, and
in solution digestion [10,11] in the latter. Although used by the great majority of proteomic
investigators, both these conventional digestion procedures need many steps, complex sample handling
and exceedingly long digestion times, which all limit high-throughput protein identification. Therefore,
new flexible and efficient tools are an urgent priority, in order to satisfy the demand for high-speed
analysis. In the last years, we have assisted to a huge increase of non-conventional digestion strategies
designed to overcome the major drawbacks encountered in conventional protein digestion procedures.
Among promising non-conventional procedures, irradiation by microwaves [12,13] or ultrasound [14],
applied both to in gel and in solution protein digestion, have shown a dramatically improved efficiency
in speed and sensitivity of proteolysis. Recently, successful in solution digestions of both single
proteins and complex protein mixtures were achieved in 60 s using pressure cycling technology in the
range of 5 to 35 kpsi [15]. Among the non conventional methods, the use of high-concentration trypsin
immobilized onto solid support has recently captured the attention of many research groups, because
these systems can significantly speed-up digestion, due to the possibility to work with enzyme-tosubstrate (E/S) ratios higher than in conventional digestion [16,17]. Enzymes for protein digestion can
be immobilized in different ways onto different kinds of favorable support (polymers, silica, porous
silicon, mesoporous silica, agarose, nanofibers, etc.) in various formats or devices (beads, cartridges,
capillaries, columns).
The so called in column digestion constitutes a new trend in current proteomics [18]. Proteolytic
enzymes, usually trypsin, are chemically immobilized and a protein solution is infused and digested
through the column. Most notably, immobilization on columns is conceived inside microfluidic
systems [19,20]. The development of microfluidic enzymatic reactors, coupled on-line with mass
spectrometers for proteomics applications, has demonstrated to be advantageous [21,22], for the
possibility of direct coupling with LC and MS [23], which eliminates possible sample loss or
contamination observed in manual handling. Therefore, protein digestion based on enzyme
immobilized into microfluidic devices, through the integration of analytical processes into MS
platforms, not only provides the rapid identification of proteins, but has also the great potential to
contribute to a substantial improvement in overall sensitivity.
Most notably, trypsin is immobilized through covalent bonds. However, in order to covalently
immobilize a protease to a specific support, many reaction steps are generally required. More than
chemical immobilization, physical adsorption into a favorable support could dramatically simplify the
experimental procedure. Recently, in such systems indeed, the immobilization of protease is
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accomplished via physical adsorption [24-31]. Mesoporous silica (MPS) materials constitute optimal
supports for physical entrapment of enzymes. The use of proteolytic enzymes, in particular trypsin,
immobilized on mesoporous materials has increasingly captured the attention of several research
groups, because it could turn highly favorable for high-throughput proteomics. We report herein an
overview of the procedures for in situ proteolysis of single proteins or complex protein mixtures, with
a special focus on MPS materials used as “nanobiocatalysts”. The challenging efforts for designing
such systems, aimed at mimicking the biochemistry of living cells, are reviewed. Potentials, limitations
and challenges of this branch of enzymatic catalysis, which we indicate as in mesopore digestion, are
discussed, in relation to its suitability for high-speed and high-throughput proteomic analysis.
2. Ordered Mesoporous Silica
According to IUPAC nomenclature, porous materials are divided into three classes: microporous
materials with a pore diameter below 2 nm, mesoporous materials with a pore diameter between 2 and
50 nm, and macroporous materials with a pore diameter above 50 nm. Ordered MPS should not be
confused with porous silica gel, which is commonly used as a stationary phase for chromatographic
separations. The surface areas of commercially available chromatographic grade silicas are generally
less than 500 m2/g, which is high relative to most materials [32]. Moreover, the wide range of pore
sizes in silica gels makes their application in shape selective catalysis unfeasible [33]. Quite different,
for their structural properties, are ordered MPSs characterized by regular arrangement of mesopores
with narrow pore size distribution, high specific surface areas (up to ca. 1500 m2/g) and pore volumes
(up to ca. 1.5 cm3/g), which render them ideal candidates as hosts for biomolecules [34]. In fact the
high surface area allows a great accessibility to molecules; moreover, the regularity of porous arrays
makes an effective control of molecular adsorption based on size easily feasible.MPSs, discovered for
the first time by Mobil oil researchers in 1992, were synthesized by the hydrolysis and condensation of
inorganic precursors (the sol-gel process) in the presence of surfactant micelles (templates) [35,36].
The possibility of synthesizing mesostructured silica materials with regularly sized pores, templating
them by the mean of supramolecolar aggregate of surfactants, represents a starting point for the design
of functional nanostructured materials. A synthetic route which is generally used for the preparation of
these materials is described below and shown in Scheme 1. Briefly, during the synthesis, the long chains
of the surfactant molecules organize into micellar liquid crystals, which serve as templates for the
formation of mesopores. When the silicate is added, it deposits itself around the micellar phase. In
many cases a “cooperative self-assembly” can take place in situ between the templates and the mineral
network precursors, leading to organized architectures. Then, the aggregates of surfactant are removed
by calcinations/extraction, generating well-ordered mesopores, separated by amorphous silica walls.
While tetraethylorthosilicate (TEOS) is commonly used as the source of silicate [37-40], a great
variety of surfactants with different alkyl chain length have been employed in order to both achieve
different geometric mesostructures and modulate pore size [41-43]. Beck et al. were able to tailor the
pore size from 1.5 to 4.5 nm by varying the chain length of quaternary trimethylammonium cations
between 8 and 18 carbon atoms. The addition of organic molecules such as 1,3,5-trimethylbenzene [36] or alkanes [44] allowed the increase in pore size up to 10 nm.
Molecules 2011, 16
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Scheme 1. The synthetic route followed to obtain mesoporous silica materials. Surfactant
molecules organize themselves, with their long chains, into micellar liquid crystals which
serve as templates for the formation of mesopores. Then, when the silicate is added, it
deposits itself around the micellar phase. A “cooperative self-assembly” takes place in situ
between the templates and the mineral network precursors, leading to organized
architectures: subsequent aggregate removal, for instance by calcinations or extraction,
generates well-ordered mesopores, separated by amorphous silica walls.
The most exploited and investigated MPSs are the M41S family members [Mobil Composition of
Matter (MCM)-41 MCM-48, MCM-50] and the Santa Barbara Amorphous (SBA)-n type materials
(SBA-1, SBA-3, and SBA-15) [45]. SBA materials show generally larger pore sizes (5–30 nm) in
comparison to MCM (1.5–10 nm). In particular SBA-15 is commonly used because its uniform
morphology can be easily controlled during the synthesis and for its exceptional capability in
biomolecules adsorption and desorption [26].
Besides the possibility of tailoring both the shape and the size of the pores, it is also possible to
modify the surface properties by introducing chemical functionalities, in order to provide specific,
desirable features. Derivatization can be performed by post-synthesis (grafting methods) or by direct
synthesis (co-condensation) [46,47]. In the former procedure, silica surface is functionalized through
silylation reactions which take place on isolated and geminal silanols, using trichloro- or
trialkoxyorganosilane and silylamines as organic precursors. In the latter, a co-condensation of
siloxane and organosiloxane precursors occurs in the presence of the corresponding structure-directing
agent. Although the co-condensation method leads to a more homogeneous surface coverage of
organic groups on silica walls, better defined and hydrolytically more stable products are obtained
when the grafting method is used [48].
MPS materials, with ordered pore structure, high surface area and high concentration of hydroxyl
groups on the surface, have captured considerable interest for a diverse range of applications, such as
catalysis, filtration and separation, molecular collection and storage, nanofluidics, medical imaging,
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drug delivery and sensors. We refer the reader to in dept literature on these topics here and below [49-51].
Recently, our group has explored a new application of MPS materials for proteomic analysis [52]. We
demonstrated that MPS materials are able to harvest peptides from plasma, serum and other bodily
fluids by the means of nano-sized porous channels with high surface area, which exclude large size
proteins by a cutoff mechanism. Extracted peptidome is then profiled and analyzed by Matrix Assisted
Laser Desorption Ionization-Time of Flight (MALDI-TOF) MS. The possibility to reveal complex
fingerprints by this material platform represents a direct avenue for biomarker discovery in readily
accessible body fluids [53,54].
Another proteomic application of MPS materials was pioneered by researchers of Fudan University
in 2005 [25]. They demonstrated that MPSs can be used as efficient nanoreactors for protein digestion.
However, the concept and the application of mesoporous materials as “natural microreactors” to
accommodate catalyst entities such as metals, metal oxide nanocomposite, semiconductors, carbon
materials and so forth were already known since the late ’90s [55,56].
The discovery of mesoporous materials has opened new horizons and opportunities to
heterogeneous catalysis. The high surface areas and the ordered periodicity of the pores, with
controllable dimensions and morphology, render mesoporous silicate optimal supports for a great
variety of catalysts, from small catalysts such as metals, metal complexes, metal oxides to large
molecule catalysts such as enzymes. In conclusion, due their above described features, MPSs offer a
broad range of applications in many fields; in particular in the last years MPSs are increasingly
exploited for proteomic analysis both in peptidome profiling both in assisted protein digestion. In the
next section we will mainly highlight the use of ordered MPSs as supports for enzyme catalysts.
3. Mesoporous Silica for Supporting Enzyme Catalysts
Enzymes are highly selective biological catalysts, which are essential in living systems for their
exquisite regio-, stereo- and substrate-specificity. Therefore, enzymes have found wide application in
medicinal chemistry, organic synthesis and biotechnology, etc. However, the use of enzymes is subject
to a number of limitations and drawbacks. For example, with few exceptions, enzymes are relatively
expensive and, therefore, disposing of them after a single cycle of production is not economical.
Moreover, enzymes have evolved to perform catalysis in very mild conditions, unless they are purified
from rare extremophile organisms (which makes their production even more expensive). An obvious
consequence is the general instability of the majority of enzymes towards heat, organic solvents, acids
or bases, etc., which limits their use in industrial applications, in which such harsh conditions are often
the norm [57]. One possible solution to these issues is to immobilize the enzyme on a appropriate
support. Not only does this facilitate the recovery and reuse of the enzyme, but in many cases,
immobilized enzymes show a higher stability than free species. Therefore, a number of methods for the
immobilization of enzymes, such as attachment to prefabricated carriers (by covalent or ionic binding,
adsorption, etc.) or cross-linkage and entrapment (encapsulation), have been developed [58].
The performances of MPS substrates as inorganic support for proteins and, in particular, enzymes
have been extensively studied. This field of research was opened by a report dating back in 1996, in
which Diaz and Balkus studied the properties of a mesoporous material, MCM-41, as support for the
adsorption of the four enzymes cytocrome c (Cyt c), trypsin, papain and peroxidase [59]. This
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pioneering work explored many of the main issues of the field. In particular, the influence of the
protein size, relative to the material pore diameters, on the support adsorption capacity was first
demonstrated. The same authors also explored the issue of enzyme leakage by reducing the pore
diameter through silanation after enzyme adsorption. Finally, the preservation of catalytic activity of
the adsorbed trypsin was also demonstrated [59]. Since then, over 150 articles have appeared in the
literature, which have explored all aspects of protein-mesoporous material interaction, including
relative size of the mesopores and the protein, mesoporous material particle size and morphology,
influence of the isoelectric points of mesoporous silicates and protein on the adsorption process,
functionalization of the silicate surface, optimization of the conditions for immobilization and the
nature of the interactions between the proteins and the support. Of fundamental interest are the issues
of preservation of enzymatic activity after immobilization and, in particular for industrial application,
of maintenance of enzymatic activity under harsh conditions (heat, organic solvents, acids or bases)
and of reusability and enhanced stability of the immobilized proteins. All these issues have been
summarized in excellent reviews [58,60] and will not be repeated here. In summary, over 60 different
proteins were immobilized on mesoporours materials of various kind, over 50 of these proteins
were enzymes and the catalytic activity of the immobilized enzyme was demonstrated with few
exceptions [60,61-79]. Studies comparing the catalytic activity of the free enzyme to that of the
immobilized form were published for over 30 different enzymes. Interestingly, the catalytic activity of
the immobilized form was determined to be higher than that of the free enzyme in 20 cases. The
immobilized form was reported to be less active than the free enzyme in 13 cases; however, in this
smaller group, the decreased catalytic activity was compensated by an enhanced stability in 10 cases,
seven of which with the additional advantage of the demonstration of the re-usability [60,61-79]. It can
therefore be concluded that not only the use of mesoporous materials as enzyme support is becoming
more and more popular in the scientific community, but these materials are beginning to demonstrate
very interesting potential applications in biocatalysis.
4. Mesoporous Silica as Supports for Protein Digestion in Proteomics
4.1. General Overview of the Field
In principle, an enzyme accelerates a specific biochemical reaction by establishing interactions with
the substrates inside its structurally pre-organized active-site. In such an environment, binding
interactions with the transition state are favored over the ground state [80].
Among other functions, trypsin is a digestive enzyme which cleaves polypeptide chains at
positively charged arginine/lysine residues [81]. In proteomics, this specificity offers several
advantages. In fact, lysine and arginine are presents in proteins primary sequences approximately every
10–12 aminoacids; consequently, tryptic fragments with an average size of 800–2,000 Da are
generated, which can be measured with modern mass spectrometers with high accuracy. In addition, in
tandem mass experiments, a favored homogeneous fragmentation of tryptic fragments occurs by
collision activated dissociation [18]. Trypsin belongs to the family of serine proteases, which covers
one third of all known proteolytic enzymes and it is so named due to the presence of a serine residue
(Ser) in the enzyme active site [82]. The catalytic triad of Asp 102, His 57, and Ser 195 residues is
Molecules 2011, 16
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responsible of the hydrolysis of the peptide bond. In particular, the nucleophilic hydroxyl O atom of
Ser 195, utilizing His 57 as a general base (stabilized by Asp 102), attacks the carbonyl of the amide
bond in the substrate, to give an oxyanion tetrahedral intermediate, stabilized by a positively charged
pocket (oxyanion hole) within the active site. After the collapse of the tetrahedral intermediate, an
acyl-enzyme intermediate and a N-terminus free polypeptide fragment are generated. The attack of a
water molecule to the acylenzyme intermediate forms a second tetrahedral intermediate, which
collapses generating new C-terminus in the substrate [83]. Trypsin, with its highly efficient fold that
couples catalysis and regulatory interactions, is an extraordinary and great machine, considering the
nanoscopic scale on which it can distinguish structural substrate rearrangement. As others enzymes,
trypsin exhibits high catalytic activity and selectivity under mild conditions, but it is difficult to handle
and it is susceptible of inactivation by pH changes, organic solvents and high temperature. Indeed high
temperature and long incubation time may lead to deamidation, transpeptidation, metionine and cysteine
oxidation and non specific cleavage. Furthermore, autolysis may also be observed [18]. Therefore, in
order to avoid the appearance of interfering autolysis peptides, low trypsin-to-substrate ratio has to be
kept. As consequence, the kinetic is slow and digestion requires several hours to proceed. Additionally,
in current proteomics workflows, where complex biological protein mixtures are completely digested,
another drawback may be encountered due to low-micromolar substrate concentrations. As just
anticipated above, a porous support can offer both a robust physical-chemical environment for trypsin
and thermal stability, enabling it to be used in cyclic operations.
The escalation of proteomics, concomitantly with the urgent demand in term of bio-analytical and
bio-chemical tools, has merged different and separated research areas into a multidisciplinary
approach. Remarkable examples of this trend are given by the great contributions brought to
proteomics from organic, analytical and material chemistry. Material sciences in particular offer to
proteomics not only a vast arrays of chromatographic phases for fractionation and separation purposes,
but also nanotextured surfaces for the enhancement of proteomic analysis via MS [54,84]. Over the last
decade, MPSs have been increasingly studied as support for enzyme immobilization for their potential
applications in biocatalysis [60]. Specifically, the interest to catalyze proteolysis by MPS supports is
matured only in the last six years, maybe due to the urgent demand of new strategies for
high-throughput proteomic analysis. Trypsin, confined in the inorganic nanospace of MPS, provides
increased cleavage efficiency. The increased bioactivity of trypsin confined into a mesoporous channel
over the free enzyme may be due a remarkable local enzyme–substrate enrichment within the
nanopores as reported in different studies in this field [25-27,29-31].
4.2. Substrate Pre-Adsorption
The first example of “in mesopore” protein digestion using MPS materials was reported by
Fan et al. [25]. The authors demonstrated the highly efficient proteolysis carried out on the enzymesubstrate model trypsin-myoglobin, using SBA-15 as a nanoreactor. Myoglobin (see Table 1 for MW,
pI and protein dimension) was firstly adsorbed into SBA-15 mesopores, then proteolysis occurred after
the addition of trypsin (Scheme 2a). In particular, in a first step, the adsorption of myoglobin was
accomplished in acidic conditions. Then the beads were centrifuged and washed before the addition of
the enzyme in an E/S = 1:2. In the last step, the beads were centrifuged and washed, then incubated in
Molecules 2011, 16
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aqueous bicarbonate solution (pH = 8.2). Therefore, the proteolysis of the entrapped myoglobin was
activated by changing the pH of the buffer from acidic to basic conditions. In this last step, the
digestion products were released from the SBA-15 meso-reactor and subsequently analyzed by
MALDI-TOF MS. In order to monitor the efficiency of the digestion, protein identification was
performed by MALDI-TOF MS, based on peptide mass fingerprinting (PMF). As control, an
experiment was also carried out without MPSs, with free trypsin in solution, using the same
experimental conditions of the in mesopore digestion. Interestingly, in this first study, the authors
investigated how different factors (substrate-enrichment, surface-character and macrostructure) could
affect the efficiency of the in mesopore digestion. MPS SBA-15 materials, with one-dimensional
mesochannels packed in a two-dimensional hexagonal structure, of different particle size and surface
character, were examined. Among the different samples investigated, SBA-15-SH, the thiol-functionalized
material, with a pore diameter of 8 nm, demonstrated the highest catalytic efficiency: Eight tryptic
peptides, generated from the in mesopore myoglobin proteolysis, were analyzed by MALDI-TOF MS,
resulting in a sequence coverage of 58% in about 10 min after trypsin addition to the beads. On the
contrary, in the control experiment with free trypsin, only three peptide matches were obtained in 12 h
with a sequence coverage of 27%. The increased efficiency of proteolysis inside the nanoporous silica
beads over the one performed with conventional in solution procedure was ascribed to the highly
efficient enrichment of the substrate and of the protease inside the porous support. Other detailed
conditions for this model system are given in Table 2.
Table 1. Molecular weight (MW), isoelectric point (pI)and size of model proteins used for
“in mesopore” digestion.
Protein
Myoglobin
Cytochrome c
Bovine Serum Albumin
Ovalbumin
Conalbumin
Lysozyme
Trypsin
a
MW (Da)
17000
12384
66400
42700
76000
14388
23400
pI
7.0
10.0
4.7
4.9
6.0
10.8
10.5
Dimension (nm)
2.1 × 3.5 × 4.4 a
2.6 × 3.2 × 3.3 a
5.0 × 7.0 × 7.0 a
7.0 × 4.0 × 5.0 b
5.0 × 5.6 × 9.5 a
1.9 × 2.5 × 4.3 a
ca. 3.8 b
protein dimensions from reference [34]; b protein dimensions from reference [60].
Later, the some group, optimized the conditions of their previous protocol, obtaining 19 tryptic
peptides with 98% of sequence coverage of myoglobin in 15 min after the addition of trypsin [26].
They varied the previous procedure by increasing the incubation time for substrate adsorption inside
MPS, by eliminating the washing steps after the substrate adsorption into MPS and by increasing the
digestion time. The elimination of washing step was adopted in order to reduce substrate loss.
Additionally, they used SBA-15 material with a pore diameter of 7 nm. The MALDI-TOF MS analysis
revealed 19 myoglobin tryptic fragments (corresponding to a sequence coverage of 98%) 15 min after
the addition of trypsin. On the contrary, in solution digestion performed in the same time span,
originated only few myoglobin fragments, unsatisfactory for unambiguous protein MS identification.
The sequence coverage of myoglobin increased to 67% only after an overnight digestion. As the
authors pointed out, with their procedure it was possible to effectively concentrate a diluted
Molecules 2011, 16
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protein/substrate solution through the confinement into the nanospace of MPSs, prior to digestion. The
increased protein concentration prior to digestion improved the enzyme kinetics, while, in absence of MPS,
the in solution digestion took place in more diluted conditions for both the substrate and the enzyme.
Scheme 2. Two common workflows for in mesopore protein digestion procedure. (a). The
substrate is adsorbed from solution into a solid mesoporous support. The enzyme is then
added and adsorbed in the mesopores. The substate is digested inside the mesopore, then
peptidic fragments are released from the support to the bulk solution and analyzed by MS;
(b). The enzyme is adsorbed from solution into a solid mesoporous support. The substrate
is then added and adsorbed into the mesopores. Digestion occurs inside the mesopore, then
peptidic fragments are released from the support to the bulk solution and analyzed by MS.
Table 2. A comparison of mesoporous materials performances for in mesopore protein digestion.
MPS
Material
(mg)
SBA-15-SH
(2.5)
SBA-15
(2.5)
FDU-12
SBA-15
MCF
(2.5)
Pore size
(nm)
Substrate
(amount digested)
E/S
(w/w)
Digestion
time
8
Myoglobin (50 μg)
1:2
10 min
7
Myoglobin (179 ng)
Cyt c (124 ng)
Myoglobin (50 μg)
Myoglobin (50 μg)
Myoglobin (50 μg)
Ovalbumin (45 μg)
/Conalbumin (15 μg)
/Cyt c (1 μg)
Nuclear proteins mouse
liver cell (10 μg)
1:2(2:1)
1:2(2:1)
1:2
1:2
1:2
1:2
15 min
15 min
15 min
15 min
15 min
15 min
1:8
30 min
17 a
8
15 b
N of
prot.frag
(S/N)
8 (70)
19 (20)
14 (20)
12 (>80)
NR(>80)
NR(>80)
Seq.
Cov.
Ref.
58%
[25]
98%
84%
84%
31%
22%
42%
26%
58%
[26]
[27]
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Table 2. Cont.
MPS
Material
(mg)
HMS(CNS)
(1)
SBA-15 SH
(3)
Pore size
(nm)
Substrate
(amount digested)
E/S
(w/w)
Digestion
time
18
Cyt c (600 μg)
Myoglobin (600 μg)
Cytoplasm of human
liver tissue (5 μg)
Cyt c/BSA c
Lysozyme/BSA d
Myoglobin/BSA d
Human serum (150 μg)
Myoglobin (20)
Myoglobin (18)
Myoglobin (16.9)
1:3
1:3
1:3
20 min
20 min
20 min
1:15
1:15
1:15
1:15
1:3
1:3
1:3
10 min c
5.7
N of
prot.frag
(S/N)
11 (NR)
12 (NR)
8 (NR)
Seq.
Cov.
Ref.
63%
NR
[29]
46%
[30]
60 min
1 min
1 min
1 min
SBA-15 (1)
4.3
7 (>50)
43%
[31]
SBA-15 (1)
6.3
2 (>50)
17%
[31]
SBA-15
4.3
7 (>50)
60%
[31]
APTES (1)
SBA-15
4.4
Myoglobin (18)
1:3
1 min
21 (>50)
100%
[31]
AAPTES (1)
a
three-dimensional interconnected nanopore networks in which ultra-large nanopores (27 nm) are
connected by expanded entrances (17 nm); b disordered mesocellar foams with large cavities
(27 nm) and wide entrance (15 nm); c the number of proteolytic fragments and sequence coverage
is referred to Cyt c (in a binary system of Cyt c and BSA); d the number of proteolytic fragments
and sequence coverage is reported at 10 min; only for the pair Cyt c and BSA, anywhere for 1 hour
of digestion the number of proteolytic peptides and the coverage is reported for all three pairs of
proteins [30]; NR: not reported; Cyt c: Cytochrome c.
With the same procedure described in the above mentioned studies and illustrated in Scheme 2
panel a, the same research group from Fudan University investigated the efficiency of a newly
synthesized mesoporous silica material FDU-12 [85,86] as meso-reactor for proteolysis [27]. The main
feature of FDU-12 was the highly ordered unique three-dimensional nanostructure in which ultra-large
nanopores (27 nm) are connected by expanded entrances (17 nm). The authors demonstrated the
efficiency of FDU-12 nanoreactor over in solution digestion, not only for individual proteins, but also
for protein mixtures. The conditions and results are reported in Table 2. Interestingly, in this study the
Authors reported the first example of in mesopore digestion of a real biological sample. In fact, a
nuclear protein fraction isolated from mouse liver cells was digested inside the FDU-12 reactor. As
control experiment, an in-solution digestion was performed on the native protein extract in the same
conditions used for the nanopore reactor, i.e., without pre-denaturation. From micro-LC MALDI-TOF/TOF
analysis more peptide signals from in-mesopore digestion were detected in comparison to that from
in-solution digestion. In fact, 98 proteins were confidently identified from the in-mesopore tryptic
profile compared to only six proteins unambiguously identified from the in solution digestion. This
superior efficiency of proteolysis within MPS nanopores without pre-denaturation over the in solution
proteolysis (whose procedure normally includes a denaturation step) implies that, when the substrate is
adsorbed inside the mesopores, it may undergo to an unfolding process making its cleavage sites fully
accessible to the enzyme. The putative interaction between basic aminoacid residues of the protein
Molecules 2011, 16
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substrate with the silanol groups present on the MPS walls may trigger the unfolding process.
Remarkably, it is worth mentioning, in this study, the exploration of some mechanistic aspects of
in mesopore digestion, in particular the postulated substrate unfolding within mesopores was supported
by experiments based on Raman spectroscopy. Interestingly, myoglobin confined in the mesospace of
the FDU-12 material showed a Raman spectrum very similar to that of myoglobin after thermal
denaturation. From the analysis of these experimental evidences, the authors hypothesized in the
“reagent-free structural unfolding” the basis for an effective entrapment, assumed as the key element
of an efficient proteolysis. Additionally, a structural factor was also found to effectively contribute to
proteolysis. In fact, the authors compared the results of in mesopore proteolysis (in term of sequence
coverage) for three different mesostructured materials: FDU-12, SBA-15 [87], and MCFs [88]. As
illustrated in Table 2, for the same amount of myoglobin substrate with the same E/S and in the same
experimental conditions, the best results were obtained for FDU-12, while only 31% and 22% of
myoglobin sequence was covered from the proteolytic peptides generated from SBA-15 and MCFs
assisted proteolysis, respectively. Therefore, the authors concluded that the enhancement of proteolytic
activity in FDU-12 over SBA-15 and MCFs was due to their structural differences. In particular, the
well organized cavities of FDU-12 facilitated the mass diffusion and transportation through the 3D
interconnected mesopores. This process was instead less favored in the 2D hexagonal packing 8 nm
channels of SBA-15 or even hindered in the disordered mesocellar structure of MCFs.
In conclusion, in this subsection different examples of in mesopore digestion have been reported in
which substrate adsorption is performed before enzyme addition. SBA-15 and FDU-12 materials (with
larger pore size) used in the above described experiments revealed their excellent ability in enriching
protein substrates, thus facilitating their digestion inside their inner surface after enzyme diffusion. In
particular SBA-15, opportunely functionalized with thiol groups, improved its adsorption efficiency
towards substrate, while the larger pores in FDU-12 material, combined with its unique three
dimensional mesostructure, were demonstrated to be useful for adsorption and consequent digestion of
complex protein mixtures.
4.3. Enzyme Pre-Adsorption
The above described mesoporous reactors for protein digestion are based on the procedure
illustrated in Scheme 2 panel (a) i.e., substrate adsorption into mesopores, followed by enzyme
addition. On the contrary, we will describe in the next pages a similar procedure with a slightly
modified workflow based on enzyme immobilization by physical adsorption inside the MPS materials
before substrate addition (Scheme 2 panel b).
Quiao and colleagues proposed a nanoporous reactor for efficient proteolysis based on cyanofunctionalized mesoporous silicate (CNS) with a pore diameter of 18 nm [29]. Their preliminary data
revealed a high adsorption capacity of CNS for trypsin (18 micromoles per g) [89]. They estimated
CNS as a good support for trypsin immobilization, substrate capture and in situ proteolysis. Two
models proteins, Cyt c and myoglobin (Table 1), but also a complex biological sample, extracted from
the cytoplasm of human liver cells, were enzymatically digested inside the CNS-nanoreactor. In order
to assess the enzyme loading necessary to guarantee the optimal substrate digestion and identification,
different amounts of trypsin were immobilized on the mesoporous support. The immobilization of
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enzyme on CNS was performed by mixing the trypsin solution with mesoporous beads suspension in
phosphate buffer at pH = 6.5, for 16 h. At pH 6.5, trypsin (pI = 10.5, Table 1) is positively charged,
therefore a favorable interaction with the negatively charged CNS walls of mesopores was
hypothesized by the authors. The CNS-trypsin particles were then separated by centrifugation, washed
with ammonium bicarbonate (pH 7.8) and then immersed for 20 min in the substrate solution (different
concentration from 2 up to 200 ng mL−1 in ammonium bicarbonate at pH = 7.8), assuring an E/S of
1:3. When the extract from human liver cells cytoplasm was digested, the protein concentration in the
solution was 1 μg μL−1. For control experiments, the conventional in solution digestion was performed
with E/S of 1:30, denaturing the substrate at 100 °C for 5 min in ammonium bicarbonate buffer, before
adding the protease, then, the solution of enzyme and substrate was incubated for 12 h at 37 °C. As
reported in Table 2, the best performances for CNS-mesoreactor were obtained when 1 mg of CNS
beads were loaded with 200 μg of trypsin and incubated in 600 μg of Cyt c; digestion led to the
identification of Cyt c by PMF (11 peptides matches by MALDI-TOF MS) with 63% of sequence
coverage. Instead, a sequence coverage of 59% was observed for Cyt c after overnight in solution
digestion. Moreover, in this study the authors found that proteolysis was still efficient also when the
concentration of proteins was reduced to 2 ng μL−1, with six peptides of myoglobin and six peptides of
Cyt c identified after a digestion time of 20 min.
Very recently, Min and colleagues [30] described a size-selective proteolysis on the mesoporous
thiol-modified SBA-15 material. In order to demonstrate the “size-selective” proteolysis, size-dependent
adsorption performance of mesoporous SBA-15-SH was proved. Four model proteins Cyt c, lysozyme,
myoglobin and BSA, with different sizes, were chosen. In Table 1 molecular weights, isoelectric
points and sizes of model proteins used for in mesopore digestion experiments are reported. Each
protein was incubated with SBA-15-SH. UV absorbance experiments were performed to determine the
amounts of protein adsorbed as function of time. Cyt c, myoglobin and lysozyme, with sizes smaller
than the pore diameter of SBA-15-SH, were rapidly adsorbed (10 min for Cyt c and lysozyme and 5 h
for the bigger myoglobin) while only a little adsorption was observed for BSA in 24 h of incubation.
Having demonstrated substrate size-dependent adsorption, the authors focused on optimizing the
conditions for enzyme immobilization. In particular, trypsin was physically adsorbed into SBA-15
MPS, modified with thiol, with pore size of 5.7 nm. The authors demonstrated that the presence of
thiol groups on SBA-15 silica walls was able to minimize the leaching of immobilized trypsin,
ascribing that to the favorable interaction between the -S-S- exposed on the surface of trypsin and thiol
groups on MPS. In a second set of experiments, the size-selective proteolysis of SBA-15-SH with
immobilized trypsin was demonstrated by digesting protein binary mixtures composed of BSA and
either Cyt c, lysozyme or myoglobin with concentration of 5 nmol mL−1 for each protein and with an
E/S ratio of 1:15 (see also Table 2 for additional details). As control experiment, the protein pairs were
also digested by free trypsin in solution. The analysis of MALDI-TOF mass spectra revealed that,
when the digestions were performed inside the mesoreactor, tryptic peptides derived from small size
proteins were always prevalent over the tryptic fragments derived from BSA. On the contrary, peptides
derived from BSA dominated the MALDI-TOF mass spectrum when free trypsin was used in control
experiments. Therefore, the authors concluded that digestion of low-MW proteins by trypsin
immobilized on SBA-15-SH was “size-dependent”, as a consequence of the size-exclusive interaction
of the mesopores against the higher MW BSA protein. Additionally, the sequence coverage of each
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protein was also analyzed by nano-LC-MS/MS. Interestingly, the authors noticed that, for prolonged
incubation times, the size-exclusive effect of the enzyme reactor toward high-MW proteins weakened.
A likely explanation of this phenomenon resides in the potential adsorption of trypsin onto the external
surface of SBA-15-SH, which therefore can digest high-MW proteins which are not totally excluded
from the adsorptive process. Consequently, shortening the digestion time constitutes a possible way to
avoid the extra digestion of high-MW proteins. Regarding to the efficiency of proteolysis for the
protein pair BSA/Cyt c, a sequence coverage of 5.27% and 45.71%, respectively, was obtained in 10 min
of incubation. These values increased to 16.64% and 74.29% after 60 min of incubation. This reactor
was also used for digestion of a complex biological sample, such as human serum. In order to
demonstrate the selectivity of proteolysis of the enzymatic mesoreactor toward the low molecular
weight proteins of serum, the tryptic peptide mixture was analyzed by nano-LC-MS/MS, after 1 h of
digestion. The data, in term of spectral count percentage versus MW of identified proteins, were
compared to those obtained using free trypsin in identical conditions. As expected, the proteins with
MW up to 40 kDa dominated the spectral counting distribution in the case of trypsin/SBA-15-SH
reactor; on the contrary MW superior to 60 kDa and mainly located in the range 60–80 kDa were
prevailing in the spectral counting distribution when free trypsin was used. Although the size-dependent
adsorption performance of mesoporous material was previously demonstrated by others for model
proteins [90] and also by our group both with models proteins [91] and with human plasma [52] and
urinary proteins [53], the originality of this study mainly resides in the “size-selective proteolysis”.
This represents a quite novel application which could be quite promising in proteomics, designed for
retrieving the peptide information from low-MW proteins selectively enhanced by the size-selective
proteolysis.
As a part of an ongoing project aimed at the development of convenient and sensitive procedures
for proteomics applications [52-54], our group has recently rationally designed and developed a new
enzymatic mesoreactor for ultrafast protein digestion [31] using the workflow shown in Scheme 2,
panel b. In particular, proteolytic fragments of myoglobin were obtained as quickly as 1 minute from
the addition of the protein to the trypsin adsorbed on N-(2-aminoethyl)-3-aminopropyl derivatized
SBA-15 MPS, with pore size of 4.4 nm, giving a 100% sequence coverage. In this study, we
investigated the effects of the pore size and of the surface modification of SBA-15 MPS on the
efficiency of both adsorption and proteolysis. To this end, MPS SBA-15 together with N-(2-aminoethyl)3-aminopropyl and aminopropyl (indicated as AAPTES and APTES, respectively) functionalized
derivatives were prepared with pore size of about 4 nm. Another support, SBA-15, with larger pore
size (6 nm), was also synthesized to monitor the effects of pore enlargement (Table 2). The design of
such mesoreactor was based on the considerations described below. In order to obtain a simple and fast
procedure for fabrication of an efficient enzyme reactor, we excluded the use of covalent bonding
between the enzyme and the support, to avoid long complicated synthesis steps. Moreover, the
chemical immobilization of trypsin into a mesoporous support, while reducing enzyme leaching from
the support itself, has been reported to modify the conformation of the enzyme with reduction of
catalytic activity [34,58]. Recent studies suggested that a stable enzyme adsorption is achieved when
mesostructured material with a pore size close to the enzyme dimension are employed [60,92].
Therefore, to obtain a well-fitting physical entrapment, we synthesized SBA-15 mesoporous supports
with pore dimensions of about 4 nm (Table 2), just slightly larger than the diameter of trypsin (Table 1).
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This ensured not only highly effective trypsin adsorption, with reduction of enzyme leaching, (which
occurs when pore dimensions are much larger than enzyme), but also shortened adsorption times from
several hours to few minutes. In ten minutes, a proteomic amount of roughly 0.3 nmol of trypsin was
adsorbed per mg of MPS for both SBA-15 APTES and SBA-15 AAPTES; the same amount was
loaded onto SBA-15 (4 nm) in 2 min and onto SBA-15 (6 nm) in only 1 min. It is worth mentioning
that, in comparison to the other published immobilization procedures, our protocol allows a quite
significant reduction of time, because we stopped the enzyme adsorption process before the
equilibrium was reached. For example the cyano-functionalized mesoreactor (with a pore diameter of
18 nm) reported by Quiao and co-workers required a loading time of 16 h at room temperature [29],
while 6 h of incubation were required for immobilizing trypsin on SBA-15-SH (pore size 5.7 nm) as
reported by Min and colleagues [30]. Our controllable “trypsin-low-charging procedure” was finely
tuned for proteomic analysis, where concentrations down to femtomoles of proteins have to be analyzed.
We observed the lowest proteolytic activity for the un-functionalized SBA-15 (6 nm), in comparison to
other SBA-15 (4 nm) materials (Table 2). In accordance with previous studies [25,29,93], a possible
explanation of this phenomenon is that, after enzyme immobilization, myoglobin diffusion and
accommodation inside the pore occurs in a more limited space, resulting in a partial substrate
unfolding, with increased exposition of accessible cleavage sites. This partial substrate unfolding
might occurs at lower efficiency when the pore diameter is larger.
In order to investigate the effects of the pore size and of the surface modification of SBA-15 MPS
on the efficiency of proteolysis, we compared the digestion products released from SBA-15 without
functional groups with those released from SBA-15-AAPTES and SBA-15-APTES, with the same
pore diameter. In the first minute, tryptic peptides released from mesoreactors lead to a sequence
coverage for myoglobin of 43%, 60% and 100% for un-functionalized SBA-15, SBA-15-APTES and
SBA-15-AAPTES, respectively. This trend was also confirmed for the following reaction time courses,
so at three minutes we observed a sequence coverage for myoglobin of 74%, 81% and 100%, for SBA-15,
SBA-15-APTES andSBA-15-AAPTES; respectively; at five minutes the sequence coverage was of
83%, 88% and 100%, in the same order. Therefore, we concluded that the presence of the amine
groups could be beneficial for the catalytic reaction. Interestingly, the length of the functional group
(APTES: 0.5 nm, AAPTES: 0.8 nm) correlates with the increased performance. It is well established
that the electrostatic stabilization of the charge in the oxyanion hole plays a crucial role in the
mechanism of serine proteases activity [83]. Interestingly, the oxyanion hole is located on the external
enzyme surface [94] and it is therefore easily accessible. Hence, it is temping to speculate that a
potential mechanism might involve a role of basic groups, in particular the longer AAPTES, in an
electrostatic stabilization of the charge in the oxyanion hole. It is worth noticing that recent studies
envisage cooperative interaction of such functional groups among organo-functionalized MPS that
give rise to enhanced catalytic activity [95].
As illustrated in all the studies reported in this section, excellent protein digestion yields were
achieved with in mesopore digestion over in solution digestion. We therefore conclude that the
elimination of several steps, generally considered essentials in the case of in solution digestion (for
instance substrate denaturation, alkylation, overnight incubation), and the rapidity of proteolysis render
the in mesopore digestion a suitable tool to simplify proteomic workflows presently in use. In addition,
mechanistic factors at the basis of in mesopore digestion have also been investigated: It is clear that the
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field is at its infancy, the few published reports illustrated here are exploratory and further in depth
studies are required to better understand and discover the great potential of MPS in proteomic analysis.
4.4. Factors Affecting in Mesopore Proteolytic Efficiency
While the effect of a functional group is specific for each chemical functionality grafted on the
support, a general issue is the relationship between pore size and enzyme dimension/activity. The
published data do not allow one to establish if in macropore digestion is more efficient than in
mesopore digestion or vice-versa. Although it is difficult to compare the performances of different
silica nanoreactors since diverse structures, geometries and experimental conditions are used,
undoubtedly, the data shown by Quian and colleagues [96] for a macroporous support such MOSF
(macroporous ordered siliceous foam) with pore size of 100 nm, also demonstrated an improvement
compared to in solution digestion. In fact, when macroporous silica materials were used as catalysts, a
better performance was observed, both for model proteins and for a complex protein mixture, in
comparison to the conventional in solution digestion. The same group has later developed a
polyfunctional device/macroreactor both for digestion and selective adsorption of glycopeptides. The
same MOSF material, with same pore size (100 nm), functionalized with boronic acid, was used to
fabricate a device. In this boronic derivatized macroporous silica material, after digestion,
glycopeptides were selectively captured based on the affinity between boronic acid and glycol groups,
while the non-specific peptides were released to the solution, or further purified by un-functionalized
MOSF and/or NH2-MOSF materials [97].
As a matter of fact, to date only a few studies have been performed to assess the factors which
regulate chemistry in nano-confinement. A great input to the field reviewed here might be provided by
an interesting kinetic model for the macropore-confined proteolysis, recently described by Bi and
colleagues [98]. To describe in macropore proteolysis, the authors assumed a multistep process,
constituted by ingress, subsequent diffusion of substrate inside the macropore, enzyme-substrate
encounter and substrate cleavage, diffusion in the macropore of peptides generated from substrate
cleavage, further cleavage and, finally, release of peptides from the macropore. On the other hand, for
conventional in solution digestion, they invoked a sequential proteolytic mechanism, in which the
enzyme hydrolyzes the amide bond of a protein or of a peptide, generating peptidic fragments which
diffuse away from the enzyme, back to the bulk solution. Therefore, the probability that the proteolytic
fragments will encounter the enzyme in the bulk solution should be much lower than that in the
macropore. In this case, the entrapment effect plays a crucial role in the kinetics of the proteolysis.
Myoglobin was used as model substrate together with angiotensin 1, and ACTH (1–14), which contain
one or two active digestion sites, respectively. Both conventional in solution digestion and in macropore
digestion were performed, maintaining the same E/S ratio to 1:30. MOSF with pore size of 100 nm,
pre-loaded with trypsin, was used for in macropore digestion. The Authors estimated a substantial
reduction in reaction time of roughly 40 times for myoglobin digested in macropore, on the basis of
proteolytic fragments (24 and seven for in macropore and in solution digestion, respectively), analyzed
by MALDI-TOF MS after 30 min of reaction. Elaborating the kinetic equations, they observed that in
addition to the entrapment (or nanoconfinement) effect, the effectiveness factor η is another key factor
that may influence the kinetics in macropores. The factor η is the equivalent of the effectiveness
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parameter used for porous media [99]; which depends from the interaction between the porous support
and the substrate. In fact, the rate of the reaction depends also by the equilibrium between substrate
diffusion and in-pore reaction. In particular, the absence of specificity between the support and the
substrate (η < 1), the overall kinetics is limited by diffusion and the enzyme reaction cannot run at its
maximum rate. On the contrary, when the affinity between porous support and substrates is strong
(i.e., electrostatic interactions, hydrophilic/hydrophobic interactions or chemical interactions), the
effectiveness factor η is ≥1, the diffusion is very fast, therefore the overall kinetics is limited by the
digestion rate. It could be interesting to assess if this model could be adapted and revisited for
in mesopore confined proteolysis.
An important issue of this field is the proteolytic activity/stability of enzyme, in this case trypsin.
The fundamental question which should be asked is: “how much physical adsorption of trypsin in a
mesoporous support stabilizes its activity?” Studies on the activity and stability of MPS-immobilized
trypsin have been done, in particular, storage stability, reusability or influence of impurities in trypsin
preparations on enzyme uptake have been reported by Goradia and colleagues [89]. In this study, the
activity of trypsin immobilized on different MPS, was found 10–20 times higher than that of the free
enzyme; moreover, MPS-immobilized-trypsin was also found stable for 4–6 weeks when stored at 4 or
25 °C. Finally, it was demonstrated that this system could be reused for up to six cycles to catalyze the
hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide.
Recently, Manyar and Colleagues investigated on the catalytic activity of porcine pepsin physically
immobilized into SBA-15, in which APTES was grafted for reducing pore openings thus minimizing
pepsin leaching [100]. The authors observed an high catalytic activity for the immobilized pepsin
similar to that of the free enzyme on the substrate Z-L-glutamyl-L-tyrosine dipeptide. On the contrary a
low catalytic activity was detected when a larger substrate such hemoglobin was used. In fact, unlike
Z-L-glutamyl-L-tyrosine, haemoglobin, with dimensions higher than the SBA-15 pores used in this
study, cannot diffuse inside the SBA-15 mesopores. Finally, the authors found that the pepsin-SBA-15
APTES bioreactor was recyclable up to four cycles without any loss of activity and could be
effectively reused. Besides trypsin and pepsin, other proteases like papain or chymotrypsin are
routinely used in proteomic protocols. Interestingly, there are reports describing the immobilization of
papain or chymotrypsin in mesoporous supports [101,102] in which the enzymatic activity was
monitored using small molecules or protein model substrates: It would be very attractive to start from
the preliminary data reported in the above cited studies and test these bioreactors for digestion of
samples to be studied in proteomic analysis. Enzymes are relatively expensive and, therefore,
discarding them after a single use is not economical. Although in the examples reviewed the amount of
enzyme is generally higher than in conventional digestion, the possibility to reuse the mesoreactor for
several cycles might amortize the high costs of the enzyme. In order to be successfully reused,
immobilized trypsin must have high activity, high stability, and high resistance to autolysis and also to
proteolysis by other proteases often found in various kinds of proteomic samples. The high
activity/stability of trypsin inside all the silica mesoreactors described in this review was proved by its
high efficiency in proteolysis (Table 2). However, only in one of the studies summarized in Table 2,
trypsin reusability for protein digestion was assessed [27]. In particular, the reusability of the protease
was tested monitoring the activity after repetitive digestions by MS analysis of peptide recovery over
16 days: Only a little decrease in activity was observed.
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A trend common to all the meso-reactors described here is the preferential choice to select physical
adsorption of enzyme/substrate, which is simple and easy to perform in comparison to long and
complex immobilization procedures. However a possible drawback encountered in this case might be
the leaching of enzyme out from the mesopores, when a weak electrostatic interaction occurs between
host and guest. In order to prevent leaching, covalent binding of the enzyme to the support has been
explored, discovering however that these procedures might lead both to a loss in enzyme activity [24,34]
and also to pore blocking effects by irreversible binding of the enzyme to pore entrance sites [103].
Although several immobilization procedure have been developed in order to limit the enzyme leakage
observed after simple adsorption, for a proteomic workflow a very simple and fast procedure would be
high desirable for digestion of single protein or complex protein mixture before MS analysis.
In conclusion, in our opinion, in the case of proteomic analysis the ease of the physical
immobilization procedure compensates for the leaching of the enzyme which, obviously, decreases the
number of rounds of reusability.
A critical issue in assessing the efficiency of such proteolytical meso-reactors, is to prove that an
effective adsorption occurs inside the nanopores, with no leaching and/or non-specific surface sorption
on outer surface of MPSs. Regarding this issue, it has been reported that when the mesopore diameter
is sufficiently large to accommodate biomolecules, proteins penetrate into the mesoporous networks,
as well as being adsorbed onto the external surface [25,29]. In fact when hexagonal mesoporous
silicate with a pore size smaller than trypsin dimension was used as mesoreactor for protein digestion,
the efficiency of proteolysis was drastically lowered in comparison to digestion performed in MPS
with pore size larger than trypsin dimension and was only a little higher compared to that in bulk
solution. To explain these data the authors hypothesized that in mesopore digestion did not occur
because the pores of hexagonal mesoporous silicate were too small for entrapping trypsin in the
internal surface [29].
Shui and colleagues observed a poor in mesopore digestion for conalbumin (pI 6) when protein
loading in FDU-12 was performed at pH of 8.5 [27]. In fact at this operative pH both protein and
silicate surface are both negatively charged, therefore electrostatic repulsion prevented substrate
entrance in the mesochannels and proteolysis did not occur. An important concern regarding this point
is the great contribution of surface derivatization to modulate electrostatic interactions between
biomolecules and MPS. Depending on the enzyme/substrate or in general protein chemical-physical
properties (idrophobicity, pI) by the means of appropriate functional groups grafted on the surface of
MPS, it is possible to improve the driving force (electrostatic interactions) for protein diffusion and
adsorption in the inner mesoporous networks thus providing a beneficial microenviroment for the
hosted biomolecule [53,58,64]. Additionally, by studying the different surface properties of SBA-15
materials, Fan and colleagues ascribed to the “surface-character effect” the different performances of
the host materials as proteolytical mesoreactors [25]. Interestingly, a poor digestion performance was
observed for the SBA-15 material in which removal of the organic template was performed by
calcination. On the contrary, the highest catalytic efficiency was observed for thiol functionalized
SBA-15 material and intermediate activity was obtained for SBA-15 material in which organic
templates were removed by ethanol extraction. A possible explanation given by the authors of this
study resides in the abundant hydroxyl groups lying on the mesochannel walls after ethanol extraction.
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It could be possible that the interaction between substrates and silanol groups accelerate the unfolding
process exposing more cleavage sites to the proteases [25].
In conclusion, although direct proof, for instance indisputable images of enzyme/substrate adsorbed
inside the pore, is still missing, several pieces of circumstantial evidence strongly suggest that the
improvement in proteolysis observed in the examples described so far can be ascribed to confinement
in the mesopores with limited effect, if any at all, contributed by non-specific surface sorption on
outside of mesoporous particles.
In the last few years the use of MPS for harvesting low molecular weight proteome from complex
biological matrix such as plasma [52-54,104-107], serum [108], urine [53], saliva and induced
sputum [109], has increased progressively. The simple and fast extraction protocols, the high
sensitivity, reproducibility and the high capacity of the MPS are surely the keys to the success of this
upcoming separation technology. Therefore we argue that in the nearly future the fruitful combination
of proteins/peptide enrichment with the in mesopore protein digestion might serve as novel useful
strategies for a deeper and high throughput proteomic analysis.
The design of MPS material to enhance protein digestion efficiency, although at its beginnings, is
quite promising and the array of possible applications in proteomics are quite broad:
(1) First of all, efficient proteolysis in real time may open new horizons to high-throughput protein
analysis. This is an essential feature, especially in the field of clinical applications of proteomics,
where a lot of specimen need to be daily and routinely analyzed.
(2) In all the mesoreactors reviewed here, (i) only a small amount (few mg) of mesoporous silica
particles is needed for digestion (Table2); (ii) proteolysis takes place in few minutes (Table 2); (iii) the
mesoreactors do not necessitate the manual sample handling and the extra steps necessary for in gel or
in solution digestion, which are time consuming and lead to the introduction of contaminants such as
human keratins.
These observations should encourage the miniaturization and the automation of the full process,
from digestion to protein identification, without intermediate steps, which should dramatically lower
even further its costs.
5. Conclusions
Confinement from molecular crowding in biological cells can both stabilize and induce order-ofmagnitude enhancements in catalysis for large molecules and proteins, compared to enzyme reactions
in solution. MPS offers an excellent platform to mimic the confinement present in such biological
systems. In fact, the presence of an ordered array of pores (whose diameter, geometry and chemical
functionalities can be finely tuned), as well as the high mechanical stability, create the optimal
environment to host enzyme and substrate in a cell-like fashion.
Since the era of mesoreactors for proteolysis is just at its infancy, there are many open questions
about the mechanisms which possibly regulate catalysis of enzymes entrapped in such supports. New
studies will be required to better address the main factors which could affect and regulate proteolytic
enzyme efficiency and stability in mesoporous support. Additional experiments are needed to better
understand which factors may enhance the stability and reusability of enzymes in mesopores.
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In our opinion a new forthcoming strategy in proteomics is shaping in the era of
nanobiotechnology. Based on the use of immobilized protease reactors, in a near future, MPS
nanobiocatalysts as “packed beads”, could be integrated into multidimensional separation systems for
automated proteomics. Due to recent progress in the field of mesostructured silica materials, new
synthetic routes will offer the opportunity to create the ideal mesospace suitable for stable enzymesubstrate entrapment. The ability to address these issues will ultimately determine how deeply material
science, in particular nanotechnology, can contribute to satisfy the urgent and challenging task of
proteomics.
Acknowledgements
We would like to thank M. Preianò for support in artwork preparation.
References and Notes
1.
Walther, T.C.; Mann, M. Mass spectrometry-based proteomics in cell biology. J. Cell Biol. 2010,
190, 491-500.
2. Yates, J.R., 3rd.; Gilchrist, A.; Howell, K.E.; Bergeron, J.J. Proteomics of organelles and large
cellular structures. Nat. Rev. Mol. Cell Biol. 2005, 6, 702-714.
3. Au, C.E.; Bell, A.W.; Gilchrist, A.; Hinding, J.; Nilsson, T.; Bergeron, J.J. Organellar proteomics
to create the cell map. Curr. Opin. Cell Biol. 2007, 19, 376-385.
4. Collins, S.R.; Kemmeren, P.; Zhao, X.C.; Greenblatt, J.F.; Spencer, F.; Holstege, F.C.;
Weissman, J.S.; Krogan, N.J. Toward a comprehensive atlas of the physical interactome of
Saccharomyces cerevisiae. Mol. Cell. Proteomics 2007, 6, 439-450.
5. Aebersold, R.; Mann, M. Mass spectrometry-based proteomics. Nature 2003, 422, 198-207.
6. Link, A.J.; Eng, J.; Schieltz, D.M.; Carmack, E.; Mize, G.J.; Morris, D.R.; Garvik, B.M.;
Yates, J.R., 3rd. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol.
1999, 17, 676-682.
7. Washburn, M.P.; Wolters, D.; Yates, J.R., 3rd. Large-scale analysis of the yeast proteome by
multidimensional protein identification technology. Nat. Biotechnol. 2001, 19, 242-247.
8. Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins
silver-stained polyacrylamide gels. Anal. Chem. 1996, 68, 850-858.
9. Wilm, M.; Shevchenko, A.; Houthaeve, T.; Breit, S.; Schweigerer, L.; Fotsis, T.; Mann, M.
Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass
spectrometry. Nature 1996, 379, 466-469.
10. Flannery, A.V.; Beynon, R.J.; Bond, J.S. Proteolysis of protein for sequencing analysis and
peptide mapping. In Proteolytic Enzymes: A Practical Approach; Beynon, R.J., Bond, J.S., Eds.;
Oxford University Press: Oxford, UK, 1989; pp. 145-162.
11. Smith, B.J. Protein sequencing protocols. In Methods in Molecular Biology, 2nd ed.; Smith, B.J.,
Ed.; Humana Press Inc: Totowa, NJ, USA, 2002; Volume 211.
12. Pramanik, B.N.; Mirza, U.A.; Ing, Y.H.; Liu, Y.H.; Bartner, P.L.; Webwe, P.C.; Bose, A.K.
Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: A new
approach to protein digestion in minutes. Protein Sci. 2002, 11, 2676-2687.
Molecules 2011, 16
5957
13. Juan, H.F.; Chang, S.C.; Huang, H.C.; Chen, S.T. A new application of microwave technology to
proteomics. Proteomics 2005, 5, 840-842.
14. Lopez-Ferrer, D.; Capelo, J.L.; Vazquez, J. Ultra fast trypsin digestion of proteins by high
intensity focused ultrasound. J. Proteome Res. 2005, 4, 1569-1574.
15. Lopez-Ferrer, D.; Petritis, K.; Hixson, K.K.; Heibeck, T.H.; Moore, R.J.; Belov, M.E.;
Camp, G.C.; Smith, R.D. Application of pressurized solvents for ultrafast trypsin hydrolysis in
proteomics: Proteomics on the fly. J. Proteome Res. 2008, 7, 3276-3281.
16. Monzo, A.; Sperling, E.; Guttman, A. Proteolytic enzyme-immobilization techniques for MS-based
protein analysis. TrAC Trends Anal. Chem. 2009, 28, 854-864.
17. Kim, J.; Kim, B.C.; Lopez-Ferrer, D.; Petritis, K.; Smith, R.D. Nanobiocatalysis for protein
digestion in proteomic analysis. Proteomics 2010, 10, 687-699.
18. Cañas, B.; Piñeiro, C.; Calvo, E.; López-Ferrer, D.; Gallardo, J.M. Trends in sample preparation
for classical and second generation proteomics. J. Chromatogr. A 2007, 1153, 235-258.
19. Wang, C.; Oleschuk, R.; Ouchen, F.; Li, J.; Thibault, P.; Harrison, D.J. Integration of immobilized
trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary
electrophoresis separations and an electrospray mass spectrometry interface. Rapid Commun.
Mass Spectrom. 2000, 14, 1377-1383.
20. Koh, W.G.; Pishkoa, M. Immobilization of multienzyme microreactors inside microfluidic
devices. Sens. Actuato. B 2005, 106, 335-342.
21. Massolini, G.; Calleri, E. Immobilized trypsin systems coupled on-line to separation methods:
Recent developments and analytical applications. J. Sep. Sci. 2007, 28, 7-21.
22. Ma, J.; Zhang, L.; Liang, Z.; Zhang, W.; Zhang, Y. Recent advances in immobilized enzymatic
reactors and their applications in proteome analysis. Anal. Chim. Acta 2009, 632, 1-8.
23. Temporini, C.; Perani, E.; Mancini, F.; Bartolini, M.; Calleri, E.; Lubda, D.; Felix, G.; Andrisano, V.;
Massolini, G. Optimization of a trypsin-bioreactor coupled with high-performance liquid
chromatography-electrospray ionization tandem mass spectrometry for quality control of
biotechnological drugs. J. Chromatogr. A 2006, 1120, 121-131.
24. Yiu, H.H.P.; Wright, P.A.; Botting, N.P. Enzyme immobilisation using siliceous mesoporous
molecular sieves. Microporous Mesoporous Mat. 2001, 44-45, 763-768.
25. Fan, J.; Shui, W.; Yang, P.; Wang, X.; Xu, Y.; Wang, H.; Chen, X.; Zhao, D. Mesoporous silica
nanoreactors for highly efficient proteolysis. Chem.-Eur. J. 2005, 11, 5391-5396.
26. Zuo, C.; Yu, W.; Zhou, X.; Zhao, D.; Yang, P. Highly efficient enrichment and subsequent
digestion of proteins in the mesoporous molecular sieve silicate SBA-15 for matrix-assisted laser
desorption/ionization mass spectrometry with time-of-flight/time-of-flight analyzer peptide
mapping. Rapid Commun. Mass Spectrom. 2006, 20, 3139-3144.
27. Shui, W.; Fan, J.; Yang, P.; Liu, C.; Zhai, J.; Lei, J.; Yan, Y.; Zhao, D.; Chen, X. Nanopore-based
proteolytic reactor for sensitive and comprehensive proteomic analyses. Anal. Chem. 2006, 78,
4811-4819.
28. Jang, S.; Kim, D.; Choi, J.; Row, K.; Ahn, W. Trypsin immobilization on mesoporous silica with
or without thiol functionalization. J. Porous Mater. 2006, 13, 385-391.
29. Qiao, L.; Liu, Y.; Hudson, S.P.; Yang, P.; Magner, E.; Liu, B. Nanoporous reactor for efficient
proteolysis. Chem.-Eur. J. 2008, 14, 151-157.
Molecules 2011, 16
5958
30. Min, Q.; Wu, R.; Zhao, L.; Qin, H.; Ye, M.; Zhu, J.J.; Zou, H. Size-selective proteolysis on
mesoporous silica-based trypsin nanoreactor for low-MW proteome analysis. Chem. Commun.
2010, 46, 6144-6146.
31. Casadonte, F.; Pasqua, L.; Savino, R.; Terracciano, R. Smart trypsin adsorption into N-(2-aminoethyl)3-aminopropyl-modified mesoporous silica for ultra fast protein digestion. Chem.-Eur. J. 2010,
16, 8998-9001.
32. Gallis, K.W.; Araujo, J.T.; Duff, K.J.; Moore, J.G.; Landry, C.C. The use of mesoporous silica in
liquid chromatography. Adv. Mater. 1999, 17, 1452-1455.
33. Zhao, X.S.; Bao, X.Y.; Guo, W.; Lee, F.Y. Immobilizing catalysts on porous materials. Materials
Today 2006, 3, 32-39.
34. Hartmann, M. Ordered mesoporous materials for bioadsorption and biocatalysis. Chem. Mater.
2005, 17, 4577-4593.
35. Kresge, C.T.; Leonowicz, M.E.; Roth, W.J.; Vartuli, J.C.; Beck, J.S. Ordered mesoporous
molecular sieves synthesized by a liquid-crystal template mechanism. Nature 1992, 359, 710-712.
36. Beck, J.S.; Vartuli, J.C.; Roth, W.J.; Leonowicz, M.E.; Kresge, C.T.; Schmitt, K.D.; Chu, C.T.W.;
Olson, D.H.; Sheppard, E.W.; McCullen, S.B.; et al. A new family of mesoporous molecular
sieves prepared with liquid crystal templates. J. Am. Chem. Soc. 1992, 114, 10834-10843.
37. Ng, L.V.; Thompson, P.; Sanchez, J.; Macosko, C.W.; McCormick, A.V. Formation of cagelike
intermediates from nonrandom cyclization during acid-catalyzed sol-gel polymerization of
tetraethyl orthosilicate. Macromolecules 1995, 28, 6471-6476.
38. Lee, Y.S.; Surjadi, D.; Rathman, J.F. Effects of aluminate and silicate on the structure of
quaternary ammonium surfactant aggregates. Langmuir 1996, 12, 6202-6210.
39. Lee, Y.S.; Surjadi, D.; Rathman, J.F. Compositional effects and hydrothermal reorganization of
mesoporous silicates synthesized in surfactant solutions. Langmuir 2000, 16, 195-202.
40. Vartuli, J.C.; Malek, A.; Roth, W.J.; Kresge, C.T.; McCullen, S.B. The sorption properties of
as-synthesized and calcined MCM-41 and MCM-48. Microporous Mesoporous Mat. 2001, 44-45,
691-695.
41. Zhao, D.; Huo, Q.; Feng, J.; Chmelka, B.F.; Stucky, G.D. Nonionic triblock and star diblock
copolymer and oligomeric surfactant syntheses of highly ordered, hydrothermally stable,
mesoporous silica structures. J. Am. Chem. Soc. 1998, 120, 6024-6036.
42. Matos, J.R.; Kruk, M.; Mercuri, L.P.; Jaroniec, M.; Zhao, L.; Kamiyama, T.; Terasaki, O.;
Pinnavaia, T.J.; Liu, Y. Ordered mesoporous silica with large cage-like pores: Structural
identification and pore connectivity design by controlling the synthesis temperature and time.
J. Am. Chem. Soc. 2003, 125, 821-829.
43. Wan, Y.; Zhao, D. On the controllable soft-templating approach to mesoporous silicates.
Chem. Rev. 2007, 107, 2821-2860.
44. Ulagappan, N.; Rao, C.N.R. Evidence for supramolecular organization of alkane and surfactant
molecules in the process of forming mesoporous silica. Chem. Commun. 1996, 24, 2759-2760.
45. Naik, B.; Ghosh, N.N. A Review on chemical methodologies for preparation of mesoporous silica
and alumina based materials. Recent Pat. Nanotechnol. 2009, 3, 213-224.
46. Wight, A.P.; Davis, M.E. Design and preparation of organic-inorganic hybrid catalysts.
Chem. Rev. 2002, 102, 3589-3614.
Molecules 2011, 16
5959
47. Vinu, A.; Hossain, K.Z.; Ariga, K. Recent advances in functionalization of mesoporous silica.
J. Nanosci. Nanotechnol. 2005, 5, 347-371.
48. Melero, J.A.; van Grieken, R.; Morales, G. Advances in the synthesis and catalytic applications of
organosulfonic-functionalized mesostructured materials. Chem. Rev. 2006, 106, 3790-3812.
49. Vallet-Regi, M.; Ruiz-Gonzales, L.; Izquierdo-Barba, I.; Gonzales-Cablet, J.M. Revisiting silica
based ordered mesoporous materials: medical applications. J. Mater. Chem. 2006, 16, 26-31.
50. Park, S.E.; Ryoo, R.; Ahn, W.S.; Lee, C.W.; Chang, R. Nanotechnology in Mesostructured
Materials; Park, S.E., Ryoo, R., Ahn, W.S., Lee, C.W., Chang, R., Eds.; Elsevier Science:
Amsterdam, The Netherlands, 2003.
51. Popat, A.; Hartono, S.B.; Stahr, F.; Liu, J.; Qiao, S.Z.; Lu, G.Q. Mesoporous silica nanoparticles
for bioadsorption, enzyme immobilization, and delivery carriers. Nanoscale 2011,
doi:10.1039/C1NR10224A.
52. Terracciano, R.; Gaspari, M.; Testa, F.; Pasqua, L.; Cuda, G.; Tagliaferri, P.; Cheng, M.M.C;
Nijdam, A.J.; Petricoin, E.F.; Liotta, L.A.; et al. Selective binding and enrichment for
low-molecular weight biomarker molecules in human plasma after exposure to nanoporous silica
particles. Proteomics 2006, 6, 3243-3250.
53. Terracciano, R.; Pasqua, L.; Casadonte, F.; Frascà, S.; Preianò, M.; Falcone, D.; Savino, R.
Derivatized mesoporous silica beads for MALDI-TOF MS profiling of human plasma and urine.
Bioconjugate Chem. 2009, 20, 913-923.
54. Terracciano, R.; Casadonte, F.; Pasqua, L.; Candeloro, P.; Di Fabrizio, E.; Urbani, A.; Savino, R.
Enhancing plasma peptide MALDI-TOF-MS profiling by mesoporous silica assisted
crystallization. Talanta 2010, 80, 1532-1538.
55. Corma, A. From microporous to mesoporous molecular sieve materials and their use in catalysis.
Chem. Rev. 1997, 97, 2373-2420.
56. Joo, S.H.; Choi, S.J.; Oh, I.; Kwak, J.; Liu, Z.; Terasaki, O.; Ryoo, R. Ordered nanoporous arrays
of carbon supporting high dispersions of platinum nanoparticles. Nature 2001, 412, 169-172.
57. Schmid, A.; Dordick, J.S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. Industrial
biocatalysis today and tomorrow. Nature 2001, 409, 258-268.
58. Yiu, H.H.P.; Wright, P.A. Enzymes supported on ordered mesoporous solids: A special case of an
inorganic-organic hybrid. J. Mater. Chem. 2005, 15, 3690-3700.
59. Diaz, J.F.; Balkus, K.J., Jr. Enzyme immobilization in MCM-41 molecular sieve. J. Mol. Catal. B:
Enzym. 1996, 2, 115-126.
60. Hudson, S.; Cooney, J.; Magner, E. Proteins in mesoporous silicates. Angew. Chem. Int. Ed. 2008,
47, 8582-8594.
61. Terrés, E.; Montiel, M.; Le Borgne, S.; Torres, E. Immobilization of chloroperoxidase on mesoporous materials for the oxidation of 4,6-dimethyldibenzothiophene, a recalcitrant organic sulfur
compound present in petroleum fractions. Biotechnol. Lett. 2008, 30, 173-179.
62. Baker, S.E.; Sawvel, A.M.; Fan, J.; Shi, Q.; Strandwitz, N.; Stucky, G.D. Blood clot initiation by
mesocellular foams: dependence on nanopore size and enzyme immobilization. Langmuir 2008,
24, 14254-14260.
63. Yu, A.; Gentle, I.R.; Lu, G.Q. Biocompatible polypeptide microcapsules via templating
mesoporous silica spheres. J. Colloid Interface Sci. 2009, 333, 341-345.
Molecules 2011, 16
5960
64. Budi Hartono, S.; Qiao, S.Z.; Jack, K.; Ladewig, B.P.; Hao, Z.; Lu, G.Q. Improving adsorbent
properties of cage-like ordered amine functionalized mesoporous silica with very large pores for
bioadsorption. Langmuir 2009, 25, 413-424.
65. Lin, J.; He, C.; Zhang, S. Immunoassay channels for alpha-fetoprotein based on encapsulation of
biorecognition molecules into SBA-15 mesopores. Anal. Chim. Acta 2009, 643, 90-94.
66. Kato, K.; Seelan, S.; Saito, T. Catalytic activity of aryl alcohol oxidase immobilized in
3D-mesoporous silicates. J. Biosci. Bioong. 2009, 108, 310-313.
67. Itoh, T.; Ishii, R.; Matsuura, S.; Mizuguchi, J.; Hamakawa, S.; Hanaoka, T.A.; Tsunoda, T.;
Mizukami, F. Enhancement in thermal stability and resistance to denaturants of lipase
encapsulated in mesoporous silica with alkyltrimethylammonium (CTAB). Colloids Surf. B 2010,
75, 478-482.
68. Sörensen, M.H.; Ng, J.B.; Bergström, L.; Alberius, P.C. Improved enzymatic activity of
Thermomyces lanuginosus lipase immobilized in a hydrophobic particulate mesoporous carrier.
J. Colloid Interface Sci. 2010, 343, 359-365.
69. Gao, S.; Wang, Y.; Diao, X.; Luo, G.; Dai, Y. Effect of pore diameter and cross-linking method
on the immobilization efficiency of Candida rugosa lipase in SBA-15. Bioresour. Technol. 2010,
101, 3830-3837.
70. Matsuura, S.; Tsunoda, T.; Shiomi, T.; Sakaguchi, K.; Hanaoka, T.; Mizukami, F. Detection of
hetero-proteins-mesoporous silica assembly by BRET. Chem. Commun. 2010, 46, 2941-2943.
71. Kato, K.; Seelan, S. Enhancing activity and stability of Burkholderia cepacia lipase by
immobilization on surface-functionalized mesoporous silicates. J. Biosci. Bioeng. 2010, 109, 615-617.
72. Wang, F.; Guo, C.; Yang, L.R.; Liu, C.Z. Magnetic mesoporous silica nanoparticles: Fabrication
and their laccase immobilization performance. Bioresour. Technol. 2010, 101, 8931-8935.
73. Yang, C.; Zhang, Z.; Shi, Z.; Xue, P.; Chang, P.; Yan, R. Application of a novel co-enzyme
reactor in chemiluminescence flow-through biosensor for determination of lactose. Talanta 2010,
82, 319-324.
74. Vittorini, M.; Dumitriu, E.; Barletta, G.; Secundo, F. Immobilization of Thermoanaerobium
brockii alcohol dehydrogenase on SBA-15. Bioprocess Biosyst. Eng. 2011, 34, 247-251.
75. Noji, T.; Kamidaki, C.; Kawakami, K.; Shen, J.R.; Kajino, T.; Fukushima, Y.; Sekitoh, T.; Itoh, S.
Photosynthetic oxygen evolution in mesoporous silica material: Adsorption of photosystem II
reaction center complex into 23 nm nanopores in SBA. Langmuir 2011, 27, 705-713.
76. Xu, L.Q.; Wen, X.H.; Ding, H.J. Immobilization of lignin peroxidase on spherical mesoporous
material. Huan Jing Ke Xue 2010, 31, 2493-2499.
77. Wu, S.; Wang, H.; Tao, S.; Wang, C.; Zhang, L.; Liu, Z.; Meng, C. Magnetic loading of
tyrosinase-Fe3O4/mesoporous silica core/shell microspheres for high sensitive electrochemical
biosensing. Anal. Chim. Acta 2011, 686, 81-86.
78. Prasad, J.; Joshi, A.; Jayant, R.D.; Srivastava, R. Cholesterol biosensors based on oxygen sensing
alginate-silica microspheres. Biotechnol. Bioeng. 2011, doi:10.1002/bit.23143.
79. Falahati, M.; Ma'mani, L.; Saboury, A.A.; Shafiee, A.; Badiei, A.R. Aminopropyl-functionalized
cubic Ia3d mesoporous silica as an efficient support for immobilization of superoxide dismutase.
Biochim. Biophys. Acta Proteins Proteomics 2011, doi:10.1016/j.bbapap.2011.04.005.
Molecules 2011, 16
5961
80. Sigala, P.A.; Kraut, D.A.; Caaveiro, J.M.M.; Pybus, B.; Ruben, E.A.; Ringe, D.; Petsko, G.A.;
Herschlag, D. Testing geometrical discrimination within an enzyme active site: constrained
hydrogen bonding in the ketosteroid isomerase oxyanion hole. J. Am. Chem. Soc. 2008, 130,
13696-13708.
81. Schechter, I.; Berger, A. On the size of the active site in proteases. I. Papain. Biochem. Biophys.
Res. Commun. 1967, 27, 157-162.
82. Di Cera, E. Serine proteases. IUBMB Life 2009, 61, 510-515.
83. Hedstrom, L. Serine protease mechanism and specificity. Chem. Rev. 2002, 102, 4501-4524.
84. Cheng, M.M.C.; Cuda, G.; Bunimovich, Y.L.; Gaspari, M.; Heath, J.R.; Hill, H.D.; Mirkin, C.A;
Nijdam, A.J.; Terracciano, R.; Thundat, T.; et al. Nanotechnologies for biomolecular detection
and medical diagnostics. Curr. Opin. Chem. Biol. 2006, 10, 11-19.
85. Fan, J.; Yu, C.Z.; Gao, T.; Lei, J.; Tian, B.Z.; Wang, L.M.; Luo, Q.; Tu, B.; Zhou, W.Z.;
Zhao, D.Y. Cubic mesoporous silica with large controllable entrance sizes and advanced
adsorption properties. Angew. Chem. Int. Ed. 2003, 42, 3146-3150.
86. Fan, J.; Yu, C.Z.; Lei, J.; Zhang, Q.; Li, T.C.; Tu, B.; Zhou, W.Z.; Zhao, D.Y. Low-temperature
strategy to synthesize highly ordered mesoporous silicas with very large pores. J. Am. Chem. Soc.
2005, 127, 10794-10795.
87. Zhao, D.Y.; Feng, J.L.; Huo, Q.S.; Melosh, N.; Fredrickson, G.H.; Chmelka, B.F.; Stucky, G.D.
Triblock copolymer syntheses of mesoporous silica with periodic 50 to 300 angstrom pores.
Science 1998, 279, 548-552.
88. Schmidt-Winkel, P.; Lukens, W.W.; Zhao, D.Y.; Yang, P.D.; Chmelka, B.F.; Stucky, G.D.
Mesocellular siliceous foams with uniformly sized cells and windows. J. Am. Chem. Soc. 1999,
121, 254-255.
89. Goradia, D.; Cooney, J.; Hodnett, B.K.; Magner, E. The adsorption characteristics, activity and
stability of trypsin onto mesoporous silicates. J. Mol. Catal. B: Enzym. 2005, 32, 231-239.
90. Yiu, H.H.P.; Botting, C.H.; Botting, N.P.; Wright, P.A. Size selective protein adsorption on thiolfunctionalised SBA-15 mesoporous molecular sieve. Phys. Chem. Chem. Phys. 2001, 3, 2983-2985.
91. Gaspari, M.; Cheng, M.M.C.; Terracciano, R.; Liu, X.; Nijdam, A.J.; Vaccari, L.; di Fabrizio, E.;
Petricoin, E.F.; Liotta, L.A.; Cuda, G.; et al. Nanoporous surface as harvesting agents for mass
spectrometric analysis of peptides in human plasma. J. Proteome Res. 2006, 5, 1261-1266.
92. Urabe, Y.; Shiomi, T.; Itoh, T.; Kawai, A.; Tsunoda, T.; Mizukami, F.; Sakaguchi, K.
Encapsulation of hemoglobin in mesoporous silica (FSM)-enhanced thermal stabilita nd resistance
to denaturants. ChemBioChem 2007, 8, 668-674.
93. Buijs, J.; Ramstrom, M.; Danfelter, M.; Larsericsdotter, H.; Hakansson, P.; Oscarsson, S.
Localized changes in the structural stability of myoglobin upon adsorption onto silica particles, as
studied with hydrogen/deuterium exchange mass spectrometry. J. Colloid Interface Sci. 2003,
263, 441-448.
94. Transue, T.R.; Krahn, J.M.; Gabel, S.A.; DeRose, E.F.; London, R.E. X-ray and NMR
characterization of covalent complexes of trypsin, borate, and alcohols. Biochemistry 2004, 43,
2829-2839.
95. Shylesh, S.; Wagner, A.; Seifert, A.; Ernst, S.; Thiel, W.R. Cooperative acid-base effects with
functionalized mesoporous silica nanoparticles: Applications in carbon-carbon bond-formation
reactions. Chem.-Eur. J. 2009, 15, 7052-7062.
Molecules 2011, 16
96.
97
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
5962
Qian, K.; Wan, J.; Qiao, L.; Huang, X.; Tang, J.; Wang, Y.; Kong, J.; Yang, P.; Yu, C.; Liu, B.
Macroporous material as novel catalysts for efficient and controllable proteolysis. Anal. Chem.
2009, 81, 5749-5756.
Qian, K.; Wan, J.; Huang, X.; Yang, P.; Liu, B.; Yu, C. A smart glycol-directed nanodevice from
rationally designed macroporous materials. Chem.-Eur. J. 2010, 16, 822-828.
Bi, H.; Qiao, L.; Busnel, J.M.; Liu, B.; Girault, H.H. Kinetics of proteolytic reactions in
nanoporous materials. J. Proteome Res. 2009, 8, 4685-4692.
Shuler, M.L.; Kargi, F. Bioprocess Engineering: Basic Concepts, 2nd ed.; Prentice Hall: Upper
Saddle River, NJ, USA, 2002.
Manyar, G.H.; Gianotti, E.; Sakamoto, Y.; Terasaki, O.; Coluccia, S.; Tumbiolo, S. Active
biocatalysts based on pepsin immobilized in mesoporous SBA-15. J. Phys. Chem. C 2008, 112,
18110-18116.
Kim, M.I.; Kim, J.; Lee, J.; Jia, H.; Na, H.B.; Youn, J.K.; Dohnalkova, A.; Grate, J.W.; Wang, P.;
Hyeon, T.; et al. Crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica:
A simple and effective method for enzyme stabilization. Biotechnol. Bioeng. 2007, 96, 210-218.
Zhao, B.; Shi, B.; Ma, R. Immobilization of papain on the mesoporous molecular sieve MCM-48.
Eng. Life Sci. 2005, 5, 436-441.
Ritter, H.; Ramm J.H.; Brühwiler, D. Influence of the structural properties of mesoporous silica
on the adsorption of guest molecules. Materials 2010, 3, 4500-4509.
Tian, R.; Zhang, H.; Ye, M.; Jiang, X.; Hu, L.; Li, X.; Bao, X.; Zou, H. Selective extraction of
peptides from human plasma by highly ordered mesoporous silica particles for peptidome
analysis. Angew. Chem. Int. Ed. Engl. 2007, 46, 962-965.
Tian, R.; Ye, M.; Hu, L.; Li, X.; Zou, H. Selective extraction of peptides in acidic human plasma
by porous silica nanoparticles for peptidome analysis with 2-D LC-MS/MS. J. Sep. Sci. 2007, 30,
2204-2209.
Qi, Y.; Wei, J.; Wang, H.; Zhang, Y.; Xu, J.; Qian, X.; Guan, Y. Improved selection of LMW
over HMW proteins from human plasma by mesoporous silica particles with external
modification. Talanta 2009, 80, 703-709.
Qi, Y.; Wu, D.; Wei, J.; Ding, K.; Wang, H.; Zhang, Y.; Qian, X.; Guan, Y. Selective extraction
of low molecular weight proteins by mesoporous silica particles with modified internal and
external surfaces. Anal. Bioanal. Chem. 2010, 398, 1715-1722.
Bouamrani, A.; Hu, Y.; Tasciotti, E.; Li, L.; Chiappini, C.; Liu, X.; Ferrari, M. Mesoporous silica
chips for selective enrichment and stabilization of low molecular weight proteome. Proteomics
2010, 10, 496-505.
Terracciano, R.; Preianó, M.; Palladino, G.P.; Carpagnano, G.E.; Foschino Barbaro, M.P;
Pelaia, G.; Savino, R.; Maselli, R. Peptidome profiling of induced sputum by mesoporous silica
beads and MALDI-TOF MS for non-invasive biomarker discovery of chronic inflammatory lung
diseases. Proteomics 2011, doi:10.1002/pmic.201000828.
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